The ability to repair DNA damage is critical to the survival of a cell, whether normal or cancerous. Upon damage, DNA repair genes and other factors are activated either to remove the damage, or, if the DNA damage is too extensive, to initiate programmed cell death. The majority of current anticancer chemotherapies rely on their ability to create unrepairable DNA lesions, leading to apoptosis/cell death. A large body of evidence proves that Nucleotide Excision Repair (NER) is an important source of resistance to chemotherapeutic reagents. Cisplatin-DNA adducts are removed by NER, the only mechanism known by which platinum-DNA intrastrand adducts are removed from DNA. Although complex (>20 proteins have so far been identified in the pathway), NER mechanisms can be summarized by five basic steps: recognition of the DNA lesion; cleavage of the damaged strand and of the lesion; excision of the damaged strand, creating a gap; synthesis of new DNA to fill the gap; and ligation of the final nick. Studies indicate that inhibition of NER would provide a rational approach to enhancing cisplatin's cytotoxicity and overcoming cisplatin resistance when the NER inhibitor is administered in combination with cisplatin. In this proposal we describe an effective, sensitive, reliable yet cost-effective assay and instrumentation for use in screening large numbers of compounds to identify commercializable inhibitors of NER. Substantial effort has been expended in search of "easy," single pot assays for DNA NER, but none has proven to be sufficiently selective, precise, and practical to lead to a reliable high throughput assay for this most important of cellular function. We believe that the only practical and reliable assay for NER will require the use of a high resolution gel electrophoresis which separates single stranded DNA fragments to basepair resolution. We have initiated a collaboration with Prof. Richard Mathies, Dept. of Chemistry, Univ. of California, Berkeley, to help with the design and implementation of a Capillary Electrophoresis (CE) apparatus that has all the properties required for HTS for DNA repair inhibitors. The system will use: microfabricated capillary array electrophoresis microplates, which will ultimately be designed to accept reaction volumes of less than 2 microliters per reaction channel; a DNA isolation procedure built into the microplate itself; and novel DNA substrates which are compatible with the fluorescence detection system designed for the microfabricated arrays. The need for small volumes for this assay arises from the relatively high cost of fluorescent DNA substrates required for assay success and the Cell-Free Extracts and cloned enzyme isolates required during the screening. Miniaturized instrumentation using a small volume system provides a major cost advantage. During Phase I, we will establish the feasibility and design characteristics of the proposed instrumentation. Phase 2 will focus on manufacturability and the successful screening of approximately 10,000 natural products as validation of the novel CE system.